The present invention relates to a filtering apparatus, enabling said operation to be performed in a minimum of time. This fast filtering apparatus is more particularly applicable to the biological field, particularly to two types of studies widely used in biochemistry and bioenergetics, namely studies of the enzymatic mechanism and measurements of ion flows through cell membranes.
The detailed study of enzymatic reactions, as well as that of the mechanisms controlling ion flows require especially designed and improved equipment, which make it possible to solve the transient events of the investigated process. Usually, the reactions catalyzed by enzymes occur at a very high speed and require methods permitting a time resolution of roughly a few milliseconds. Hitherto, advances in connection with the mechanisms of the enzymatic action have generally followed an improvement in the method used and in particular the time resolution obtained.
Thus, it is known that the duration of an enzymatic cycle is generally well below 1 second. It is generally possible to identify a relatively large number of intermediate states in a cycle. Rapid mixing or multimixing methods have been developed in the past, with a view to investigating these transient states. In principle, these methods are relatively simple, the enzyme E is mixed very rapidly (a few milliseconds) with a molecule or substrate S, the reaction then following its course and is completed during the release of the product P: ##STR1##
The analysis of the intermediate stages (in this case the state E..sup.+ S) is performed by carrying out a second fast mixing (a few milliseconds to a few seconds after the initial mixing) with a reagent Q, which stabilizes E.sup.+ S. The latter is then analyzed by conventional, but often slow biochemical methods. The enzyme is mixed very rapidly with the molecule or substrate with which it must combine before producing its modification. The object is to investigate the reaction (1).
The enzyme is brought into contact with the molecule to be investigated (or substrate S). In a second stage, the enzyme and the molecule or substrate are combined, followed by the modification of the molecule by the enzyme. The reaction is blocked at this stage with the aid of a reagent Q. Thus, it is possible to analyze the blocked (E.sup.+ S) by conventional biochemical methods, such as the aforementioned fast mixing.
It is possible to block the reaction at all stages, if there is an appropriate reagent (E.sup.+ S,E.sup.+ P, E+P). Another method consists of substituting the natural substrate S for a chemical analog (molecule having the same chemical properties, but special physical properties, which can be optically followed, e.g. fluorescence and absorptivity) making it possible to follow the progress of the reaction (1) as a function of time. The time resolution of fast mixing methods and optical methods is approximately 10 msec.
Ion flow measurements (ion flow=measurement of the flow of molecules, ions, etc entering and leaving a molecule) have in the past almost exclusively been carried out by using filtering methods, e.g. Millipore filters. Briefly, the principle of these measurements is as follows. A suspension of membranous vesicles or cells is diluted in a medium containing the substrate or molecule, whereof it is wished to investigate the ion flow which has to be transported into the cell. Generally, the substrate is labelled by a radioactive isotope. Following an incubation time of varying length, the suspension is filtered. The membranes or cells are held back by the filter and the transport is evaluated by measuring the reactivity of the filter with the aid of a scintillation counter. This method is relatively slow (minimum 2 seconds), but it is the only method which is presently available. It is consequently widely used, because flow measurements are very important in research on the physiology and energetics of cells, as well as in molecular pharmacology.